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Download 1. Types of Microscopy The Electromagnetic Spectrum 9/13/2016 Chapter 4A:
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9/13/2016 Chapter 4A: Microscopy & Microbial Classification 1. Types of Microscopy 2. Staining 3. Classification & Nomenclature 1. Types of Microscopy Chapter Reading – pp. 96-107 The Electromagnetic Spectrum 400 nm 700 nm Visible light Gamma rays UV X rays light Infrared Microwave Radio waves and Television Increasing wavelength 1012m 108m 104m 100m 103m Crest One wavelength Trough Increasing resolving power 1 9/13/2016 Scale of Magnification Diameter of DNA Ribosomes Typical bacteria and archaea Flea Pig Atoms Proteins Amino acids 0.1 nm 1 nm Mitochondrion 10 nm Scanning tunneling microscope (STM) 0.5 nm–10 nm 100 nm Large protozoan (Euglena) Chloroplasts Viruses 1 m Chicken egg Human red blood cell 10 m 100 m 1 mm 10 mm 100 mm 1m 10 m Transmission electron microscope (TEM) 10 nm–100m Atomic force microscope (AFM) 1 nm–10 nm Scanning electron microscope (SEM) 10 nm–1 mm Compound light microscope (LM) 200 nm–10 mm Unaided human eye 200 m– Light Microscopy a typical “bright field” microscope such as used in lab Oil Immersion & Light Refraction Microscope objective Refracted light rays lost to lens Unrefracted light rays enter lens Glass cover slip Immersion oil Glass cover slip Slide Specimen Microscope objective Lenses Slide Light source Without immersion oil Specimen Light source With immersion oil Different media (air, water, glass, oil…) bend light to different degrees. • i.e., have different refractive indexes • immersion oil has refraction index similar to glass, allows more light to enter the lens 2 9/13/2016 (“normal” light microscopy) Bright vs Dark Field Microscopy Light refracted by specimen Objective Light unrefracted by specimen Specimen Condenser Dark-field stop only light refracted by the specimen will enter the objective lens Dark-field stop Phase Contrast Microscopy Enhances misalignment of light waves to create contrast • reveals internal detail without staining • useful for live specimens Rays in phase Nucleus Rays out of phase Phase plate Bacterium Ray deviated by specimen is 1/4 wavelength out of phase. Deviated ray is now 1/2 wavelength out of phase. Phase contrast Differential Interference Contrast (DIC) Microscopy A variation on phase-contrast microscopy involving a more complex combination of filters and prisms. • also referred to as “Normarski Microscopy” Bacteria • creates an image with even greater detail and contrast • image has a 3-dimensional appearance as if it was illuminated from the side Nomarski 3 9/13/2016 Fluorescent Microscopy Fluorescent dyes or antibodies with a fluorescent tag stick to specific targets. Under UV light, dye fluoresces, only labeled cells or structures are seen. standard confocal Confocal Fluorescence Microscopy Only light from a given depth or plane is transmitted, “out of focus” light is excluded Electron Microscopy Electromagnetic lenses focus electron beam onto metal-stained specimen. • electron beams have very short wavelengths • allows far greater resolution than with light microscopy Transmission EM (TEM) • thin sections of specimen, highest resolution Scanning EM (SEM) • reveals surface features 4 9/13/2016 Other types of Microscopy Scanning-Tunneling & Atomic Force microscopy use special fine-tipped probes to produce highest resolution. scanning-tunneling (STM) SCANNINGTUNNELING • distance between probe and specimen determined by electron flow between them ATOMIC FORCE Plasmid DNA DNA double helix • deflection of laser aimed at probe tip produces image atomic force (AFM) 2. Staining Chapter Reading – pp. 108-111 Why the Need for Stains? Because, no matter how high the magnification or resolution, you need contrast to be able to see anything. If contrast is not sufficient in the sample or the microscopic method used, staining can provide the necessary contrast: • stains used for viewing bacteria via light microscopy are typically positively charged chromophores (basic dyes) • chromophore = “color-bearing” ion of a salt • bacteria have a net negative charge (i.e., bind positive ions) 5 9/13/2016 General Types of Stains Simple stain • dye that non-specifically stains all organisms, features Differential stain • dye that binds various structures or organisms differently Counter stain • a 2nd dye added that is a different color than original dye Negative stain • dye that stains background, not specimen Special stain • dye that specifically stains certain subcellular structures **a mordant is any chemical added to enhance a stain** Fixing the Specimen on a Slide Specimens must first be “fixed” to the slide surface before they can be stained. Spread culture in thin film over slide Pass slide through flame to fix it Air dry • generally done by smearing a sample on the slide, air drying, and passing briefly through flame to “heat fix” • specimens can also be fixed to the slide surface by chemical means Gram Staining A very common stain to distinguish 2 bacterial types: 1 2 3* 4 Gram positive • retains primary stain (due to thick peptidoglycan layer and teichoic acids) Gram negative Process: 1) 1o stain (crystal violet) • does NOT retain primary stain, only counter stain 2) mordant (iodine) 3) decolorize* (alcohol) * key step 4) counter stain (safranin) 6 9/13/2016 Acid-Fast Staining Most bacteria are not “acid-fast” (i.e., don’t retain primary stain after acid wash). • “acid fast” bacteria detected by this stain include those in the genera: Mycobacterium Nocardia acid fast non-acid fast Special Stains • “non-acid-fast” cells revealed by counter stain Flagella Stain • reveal specific subcellular structures Capsule Stain Endospore Stain 3. Classification & Nomenclature Chapter Reading – pp. 113-115 7 9/13/2016 Taxonomic Hierarchy Species Genus Family Order Class Phylum Kingdom Domain Ursus americanus (American black bear) Ursus Ursidae Carnivora Mammalia Chordata species can also be subdivided different strains Animalia Eukarya Scientific Nomenclature To avoid confusion, every type of organism must be referred to in a consistent way. The current system of nomenclature (naming) has been in use since the 18th century: • every type of organism is referred by its genus name followed by its specific epithet (i.e., species name) Homo sapiens (H. sapiens) Escherischia coli (E. coli) • name should be in italics and only the genus is capitalized which can also be abbreviated • names are Latin (or “Latinized” Greek) with the genus being a noun and the specific epithet an adjective **strain info can be listed after the specific epithet (e.g., E. coli O157:H7)** Key Terms for Chapter 4A • resolution, refraction & oil immersion • microscopy: bright & dark field, phase contrast, DIC, fluorescent, confocal, transmission & scanning EM, scanneling-tunneling, atomic force microscopy • simple, differential, counter, negative stains • mordant, chromophore Relevant Chapter Questions MC: 1-10 FB: 1-5 Labeling SA: 1, 3-6 8
